Attachment of unmodified nucleic acids to silanized solid phase surfaces

ABSTRACT

The invention relates to a simple, cost effective method for immobilizing synthetic nucleic acid molecules onto a solid support. The invention further concerns the use of such immobilized molecules in nucleic acid hybridization assays, sequencing by hybridization assays, and genetic analyses and combinatorial analyses involving nucleic acids or proteins for screening applications.

FIELD OF THE INVENTION

The invention relates to a simple, and preferably cost effective, methodfor immobilizing nucleic acid molecules onto a solid support. Theinvention further concerns the use of such immobilized molecules innucleic acid hybridization assays, sequencing by hybridization assays,and genetic analyses and combinatorial analyses involving nucleic acidsor proteins for screening applications.

BACKGROUND OF THE INVENTION

The analysis of the structure, organization and sequence of nucleic acidmolecules is of profound importance in the prediction, diagnosis andtreatment of human and animal disease, in forensics, in epidemiology andpublic health, and in the elucidation of the factors that control geneexpression and development. Methods for immobilizing nucleic acids areoften important in these types of analyses. Three areas of particularimportance involve hybridization assays, nucleic acid sequencing, andthe analysis of genomic polymorphisms.

I. Nucleic Acid Hybridization

The capacity of a nucleic acid "probe" molecule to hybridize (i.e. basepair) to a complementary nucleic acid "target" molecule forms thecornerstone for a wide array of diagnostic and therapeutic procedures.

Hybridization assays are extensively used in molecular biology andmedicine. Methods of performing such hybridization reactions aredisclosed by, for example, Sambrook, J. et al. (In: Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), Haymes, B. D., et al. (In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985))and Keller, G. H. and Manak, M. M. (In: DNA Probes, Second Edition,Stockton Press, New York, N.Y. (1993)) which references are incorporatedherein by reference.

Many hybridization assays require the immobilization of one component.Nagata et al. described a method for quantifying DNA which involvedbinding unknown amounts of cloned DNA to microtiter wells in thepresence of 0.1M MgCl₂ (Nagata et al., FEBS Letters 183: 379-382, 1985).A complementary biotinylated probe was then hybridized to the DNA ineach well and the bound probe measured calorimetrically. Dahlen, P. etal. have discussed sandwich hybridization in microtiter wells usingcloned capture DNA adsorbed to the wells (Dahlen, P. et al., Mol. Cell.Probes 1: 159-168, 1987). An assay for the detection of HIV-1 DNA usingPCR amplification and capture hybridization in microtiter wells has alsobeen discussed (Keller, G. H. et al., J. Clin. Microbiol. 29: 638-641,1991). The NaCl-mediated binding of oligomers to polystyrene wells hasbeen discussed by Cros et al (French patent no. 2,663,040) and veryrecently by Nikiforov et al. (PCR Methods Applic. 3: 285-291, 1994). Thecationic detergent-mediated binding of oligomers to polystyrene wellshas very recently been described by Nikiforov et al., Nucleic Acids Res.22: 4167-4175.

II. Analysis Of Single Nucleotide DNA Polymorphisms

Many genetic diseases and traits (i.e. hemophilia, sickle-cell anemia,cystic fibrosis, etc.) reflect the consequences of mutations that havearisen in the genomes of some members of a species through mutation orevolution (Gusella, J. F., Ann. Rev. Biochem. 55:831-854 (1986)). Insome cases, such polymorphisms are linked to a genetic locus responsiblefor the disease or trait; in other cases, the polymorphisms are thedeterminative characteristic of the condition.

Such single nucleotide polymorphisms differ significantly from thevariable nucleotide type polymorphisms ("VNTRs"), that arise fromspontaneous tandem duplications of di- or tri-nucleotide repeated motifsof nucleotides (Weber, J. L., U.S. Pat. No. 5,075,217; Armour, J. A. L.et al., FEBS Lett. 307:113-115 (1992); Jones, L. et al., Eur. J.Haematol. 39:144-147 (1987); Horn, G. T. et al., PCT ApplicationWO91/14003; Jeffreys, A. J., U.S. Pat. No. 5,175,082); Jeffreys. A. J.et al., Amer. J. Hum. Genet. 39:11-24 (1986); Jeffreys. A. J. et al.Nature 316:76-79 (1985); Gray, I. C. et al. Proc. R. Acad. Soc. Lond.243:241-253 (1991); Moore, S. S. et al. Genomics 10:654-660 (1991);Jeffreys, A. J. et al. Anim. Genet. 18:1-15 (1987); Hillel, J. et al.Anim. Genet. 20:145-155 (1989); Hillel, J. et al. Genet. 124:783-789(1990)), and from the restriction fragment length polymorphisms("RFLPs") that comprise variations which alter the lengths of thefragments that are generated by restriction endonuclease cleavage(Glassberg, J., UK patent application 2135774; Skolnick, M. H. et al.,Cytogen. Cell Genet. 32:58-67 (1982); Botstein, D. et al. Ann. J. Hum.Genet. 32:314-331 (1980); Fischer, S. G. et al. (PCT ApplicationWO90/13668); Uhlen, M., PCT Application WO90/11369)).

Because single nucleotide polymorphisms constitute sites of variationflanked by regions of invariant sequence, their analysis requires nomore than the determination of the identity of the single nucleotidepresent at the site of variation; it is unnecessary to determine acomplete gene sequence for each patient. Several methods have beendeveloped to facilitate the analysis of such single nucleotidepolymorphisms.

Mundy, C. R. (U.S. Pat. No. 4,656,127), for example, discusses a methodfor determining the identity of the nucleotide present at a particularpolymorphic site that employs a specialized exonuclease-resistantnucleotide derivative. A primer complementary to the allelic sequenceimmediately 3' to the polymorphic site is permitted to hybridize to atarget molecule obtained from a particular animal or human. If thepolymorphic site on the target molecule contains a nucleotide that iscomplementary to the particular exonuclease-resistant nucleotidederivative present, then that derivative will be incorporated onto theend of the hybridized primer. Such incorporation renders the primerresistant to exonuclease, and thereby permits its detection. Since theidentity of the exonuclease-resistant derivative of the sample is known,a finding that the primer has become resistant to exonucleases revealsthat the nucleotide present in the polymorphic site of the targetmolecule was complementary to that of the nucleotide derivative used inthe reaction. The Mundy method has the advantage that it does notrequire the determination of large amounts of extraneous sequence data.It has the disadvantages of destroying the amplified target sequences,and unmodified primer and of being extremely sensitive to the rate ofpolymerase incorporation of the specific exonuclease-resistantnucleotide being used.

Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087)discuss a solution-based method for determining the identity of thenucleotide of a polymorphic site. As in the Mundy method of U.S. Pat.No. 4,656,127, a primer is employed that is complementary to allelicsequences immediately 3' to a polymorphic site. The method determinesthe identity of the nucleotide of that site using labeleddideoxynucleotide derivatives, which, if complementary to the nucleotideof the polymorphic site will become incorporated onto the terminus ofthe primer.

An alternative method, known as Genetic Bit Analysis™ or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3' to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase. It isthus easier to perform, and more accurate than the method discussed byCohen.

Cheesman, P. (U.S. Pat. No. 5,302,509) describes a method for sequencinga single stranded DNA molecule using fluorescently labeled 3'-blockednucleotide triphosphates. An apparatus for the separation, concentrationand detection of a DNA molecule in a liquid sample has been recentlydescribed by Ritterband, et al. (PCT patent Application No. WO95/17676).

An alternative approach, the "Oligonucleotide Ligation Assay" ("OLA")(Landegren, U. et al. Science 241:1077-1080 (1988)) has also beendescribed as capable of detecting single nucleotide polymorphisms. TheOLA protocol uses two oligonucleotides which are designed to be capableof hybridizing to abutting sequences of a single strand of a target. Oneof the oligonucleotides is biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA. In addition to requiring multiple, andseparate, processing steps, one problem associated with suchcombinations is that they inherit all of the problems associated withPCR and OLA.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A. -C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA™ in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A. -C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)). Such a range oflocus-specific signals could be more complex to interpret, especiallyfor heterozygotes, compared to the simple, ternary (2:0, 1:1, or 0:2)class of signals produced by the GBA™ method. In addition, for someloci, incorporation of an incorrect deoxynucleotide can occur even inthe presence of the correct dideoxynucleotide (Komher, J. S. et al.,Nucl. Acids. Res. 17:7779-7784 (1989)). Such deoxynucleotidemisincorporation events may be due to the Km of the DNA polymerase forthe mispaired deoxy-substrate being comparable, in some sequencecontexts, to the relatively poor Km of even a correctly base paireddideoxy-substrate (Kornberg, A., et al., In: DNA Replication, SecondEdition (1992), W. H. Freeman and Company, New York; Tabor, S. et al.,Proc. Natl. Acad. Sci. (U.S.A.) 86:4076-4080 (1989)). This effect wouldcontribute to the background noise in the polymorphic siteinterrogation.

III. Methods of Immobilizing Nucleic Acids to a Solid Phase

Several of the above-described methods involve procedures in which oneor more of the nucleic acid reactants are immobilized to a solidsupport. Currently, 96-well polystyrene plates are widely used insolid-phase immunoassays, and several PCR product detection methods thatuse plates as a solid support have been described. The most specific ofthese methods require the immobilization of a suitable oligonucleotideprobe into the microtiter wells followed by the capture of the PCRproduct by hybridization and colorimetric detection of a suitablehapten. It would be desirable to have an improved immobilization methodthat could be used to bind oligonucleotides to polystyrene such thattheir capacity to be used for hybridization, sequencing, or polymorphicanalysis would be retained, and which would be rapid, convenient to useand inexpensive. The present invention provides such an improved method.

The means by which macromolecules bind non-covalently to polystyrene andglass surfaces is not well understood. Nevertheless, these adsorptionphenomena have proven to be important in the development andmanufacturing of immunoassays and other types of diagnostic tests whereone component needs to be immobilized.

Polystyrene is a very hydrophobic material because it normally containsno hydrophilic groups. Microtiter plate manufacturers have developedmethods of introducing such groups (hydroxyl, carboxylate and others)onto the surface of microwells to increase the hydrophilic nature of thesurface. Theoretically, this allows macromolecules to bind through acombination of hydrophobic and hydrophilic interactions (Baier et al.,Science 162: 1360-1368 (1968); Baier et al., J. Biomed. Mater. Res. 18:335-355 (1984); Good et al., in L. H. Lee (ed.) Fundamentals ofAdhesion, Plenum, New York, chapter 4 (1989)) (FIG. 1). In practice,some proteins do bind more efficiently to the treated hydrophilicpolystyrene than to the untreated material. Covalent binding topolystyrene, especially microtiter wells, has however proven to bedifficult, so passive adsorption remains the most commonly used methodof binding macromolecules to such wells. The term "polystyrene" may alsobe used to describe styrene-containing copolymers such as:styrene/divinyl benzene, styrene/butadiene, styrene/vinyl benzylchloride and others.

While polystyrene is an organic hydrophobic substrate, glass provides aninorganic hydrophilic surface. The most common glass format inimmunoassays is the microscope slide. Laboratory-grade glasses arepredominantly composed of SiO₂, but they also may contain B₂ O₃, Al₂ O₃as well as other oxides (FIG. 2). Interfaces involving such materialshave thus become a dynamic area of chemistry in which surfaces have beenmodified in order to generate desired heterogeneous environments or toincorporate the bulk properties of different phases into a uniformcomposite structure. Our purpose here then is to use organosilanes fortailoring surfaces with chemically reactive groups mercapto (SH) and/orepoxy.

While numerous methods for the attachment of oligonucleotides andproteins on surfaces have been described, the methods are both expensiveand time consuming. The reported covalent attachments of pre-madeoligonucleotides onto modified glass surfaces have been always usingmodified oligonucleotides in order to increase reactivity andselectivity of oligonucleotides towards surfaces. Typical modificationsinvolved the introduction of amino groups, or thio groups into 3'-and/or 5'-oligonucleotides. For example, Stimpson et al. (P.N.A.S.92:6379-6383 (1995)) reported covalent attachment of 3'-aminooligonucleotides onto epoxy silanized surfaces with acid catalysis butwith only 1/10 the density achieved in this invention. Beattie et al.(Clin. Chem. 41:700-706 (1995)) reported attachment of 3'- and/or 5'-amino oligonucleotides onto epoxy silanized surfaces under elevatedtemperature. Lamture et al. (Nucleic Acids Res. 22:2121-2125 (1994))reported the methods for attaching 3'-amino-oligonucleotides onto epoxysilanized slides under 0.1M KOH. Hetero bifunctional cross-linkages havebeen used to couple the 3' or 5'-thio-modified oligonucleotides oramino-modified onto amino-propyl silanized surfaces as reported byChrisey et al. (Nucleic Acids Res. 24: 303103039 (1996)) and Guo et al.(Nucleic Acids Res. 22: 4556-5465 (1994)). All of these reported methodshowever, require modified oligonucleotides.

The present invention describes a novel method for immobilizing nucleicacid molecules to a solid-phase by means of a covalent ether orthioether linkage. This simple, two-step method has the specificity andefficiency needed to prepare DNA arrays.

SUMMARY OF THE INVENTION

The present invention provides an improved immobilization method thatpermits the rapid, and inexpensive immobilization of nucleic acidmolecules to a solid phase. The invention allows immobilization ofoligonucleotides by incubation with a silane-containing orsilane-treated solid phase. The immobilized molecules can be used fornucleic acid hybridization assays, sequencing hybridization assays,genetic analyses, combinatorial analyses involving nucleic acids orproteins, and other screening applications such as protein-DNA bindingassays.

In detail, the invention provides a method for immobilizing a nucleicacid molecule to a solid phase, the method comprising the steps of:

(A) coating said solid phase with a silane and allowing saidsilane-coated solid phase to cure; and

(B) coupling unmodified nucleic acid molecules having either a terminal3' OH or a terminal 5' OH to said silane-coated solid phase.

The invention particularly concerns the embodiments of the above methodwherein, in step A, the silane is selected from the group consisting of3-mercapto-propyl-trimethoxysilane and 3-glycidoxypropyl-trimethoxysilane.

The invention further pertains to oligonucleotide coated surfaces usefulin genetic analysis and other screening applications such as protein-DNAbinding assays.

The invention particularly concerns oligonucleotide arrays comprisingepoxy- or mercapto-silane coated surfaces and unmodifiedoligonucleotides covalently attached to the epoxy or mercapto-silane,such arrays being useful for nucleic acid hybridization assays,sequencing hybridization assays, genetic analyses, combinatorialanalyses involving nucleic acids or proteins, and other screeningapplications such as protein-DNA binding assays. The features of thecoated surface enable standardized patterning of unique oligonucleotidesonto silane surface coatings.

The invention further pertains to simultaneous patterning of multipleDNA probes in a high density and in a variety of array formats.

DETAILED DESCRIPTION OF THE INVENTION I. The Immobilization of NucleicAcid Molecules

The present invention concerns a method for immobilizing nucleic acidmolecules onto a solid-phase. Recently, several methods have beenproposed as suitable for immobilizing an oligonucleotide to a solidsupport. Holmstrom, K. et al., for example, exploit the affinity ofbiotin for avidin and streptavidin, and immobilize biotinylated nucleicacid molecules to avidin/streptavidin coated supports (Holmstrom, K. etal., Anal. Biochem. 209:278-283 (1993)). Another method requires theprecoating of the polystyrene or glass solid phases with poly-L-Lys orpoly L-Lys, Phe, followed by the covalent attachment of either amino- orsulfhydryl-modified oligonucleotides using bi-functional crosslinkingreagents. Unlike the present invention, both methods require the use ofmodified oligonucleotides as well as a pretreatment of the solid phase;however, the present invention discloses a method to covalently bondoligonucleotides with an "unmodified" 5' or 3'--OH to a solid surface.As used herein, the term "unmodified" refers to the absence of anyrequirement for specialized reactive groups. It does not refer to theexclusion of nucleotides or oligonucleotides that have such groups, orthat are biotinylated, flouresceinated, etc.

Kawai, S. et al. describes an alternative method in which shortoligonucleotide probes were ligated together to form multimers and thesewere ligated into a phagemid vector (Kawai, S. et al., Anal. Biochem.209:63-69 (1993)). The oligonucleotides were immobilized ontopolystyrene plates and fixed by UV irradiation at 254 nm. A method forthe direct covalent attachment of short, 5'-phosphorylated primers tochemically modified polystyrene plates ("Covalink" plates, Nunc) hasalso been proposed by Rasmussen, S. R. et al., (Anal. Biochem.198:138-142 (1991)). The covalent bond between the modifiedoligonucleotide and the solid phase surface is created by a condensationreaction with a water-soluble carbodimide. The Rasmussen method claims apredominantly 5'-attachment of the oligonucleotides via their5'-phosphates; however, it requires the use of specially prepared,expensive plates. The method of the present invention departs from suchmethods, in not requiring such unstable and difficult to manipulatecrosslinking reagents.

Maskos, U. et al. describes a method to synthesize oligonucleotidesdirectly onto a glass support (Maskos, U. et al., Nucl. Acids Res.20:1679-1684 (1992)). According to this method, a flexible linker with aprimary hydroxyl group is bound to the solid support via aglycidoxypropyl silane, wherein the primary hydroxyl group serves as thestarting point for the oligonucleotide synthesis.

Numerous methods for the attachment of oligonucleotides and proteins onsurfaces have been described. The reported covalent attachments ofpremade oligonucleotides onto glass surfaces have been always usingmodified oligonucleotides in order to increase reactivity andselectivity of oligonucleotides towards surfaces. Typical modificationsinvolved the introduction of amino groups, or thio groups into 3'- and/or 5'- amino modified oligonucleotides onto epoxy surfaces. Stimpson etal. (P.N.A.S. 92:6379-6383 (1995)) discloses the generation of DNA chipsfor use in automated DNA diagnostic applications. To this effect, theStimpson article discloses the immobilization of 3' amino-linkedoligonucleotides into an organized array on a glycidoxypropyl silanetreated glass slide.

Lamture et al. (Nucleic Acids Res. 22:2121-2125 (1994)) describes theimmobilization of 3' amino modified oligonucleotides to a3-glycidoxypropyl-trimethoxysilane coated silicon wafer in the presenceof 0.1M KOH. The oligonucleotides are covalently immobilized to thesolid support by means of a secondary amine linkage. Beattie et al.(Clin. Chem. 41:700-706 (1995)) discloses the immobilization of either3' and/or 5' amino modified oligonucleotides to3-glycidoxypropyltrimethoxysilane coated glass slides at a temperatureof 60° C. Additionally, several other references generally related tothe immobilization of oligonucleotides to a solid-support. Chrisey etal. (Nucleic Acids Res. 24:3031-3039 (1996), Guo et al. (Nucleic AcidsRes. 22:45456-5465 (1994), Fahy et al. (Nucleic Acids Res. 21:1819-1826(1993), Sliwkowski et al. (Biochem. J. 209:731-739 (1983) all disclosethe immobilization of a 5' or 3' modified oligonucleotide to asolid-support.

The method of the present invention provides three distinct advantagesover other covalent attachment chemistries for oligonucleotide arraypreparation. First, while the above-identified prior art referencesrequire that the oligonucleotide be either amino or thiol modified, thepresent invention discloses a method to covalently bond oligonucleotideswith an "unmodified" 5' or 3' --OH to a solid surface. The presentinvention thus differs from conventional methods for the covalentattachment of oligonucleotides to solid phases in that it permits thecovalent attachment of "unmodified" oligonucleotides. Accordingly, thepresent invention provides several significant improvements over theprior art. In particular, the present invention provides a low-cost,stable method for the covalent attachment of "unmodified"oligonucleotides to a silanized solid-phase wherein the covalently boundoligonucleotides have wide uses in genetic and combinatorial analysisinvolving nucleic acids or proteins. Covalent attachment ofoligonucleotides onto solid phase surfaces is therefore achieved withoutmodification of oligonucleotides, thereby dramatically reducing the costand eliminating the variation in quality of modified oligonucleotides.

Second, the silanized surface of the present method provides a veryhydrophobic surface which allows oligonucleotide probe droplets to format specific and localized positions on the solid surface. Thus, forexample, multiple probes can be patterned simultaneously on the surfaceusing, for example, a robotic liquid delivery system or an ink-jetprinting technique with no cross contamination between probes, even at ahigh probe density (10,000 probes/cm²). Accordingly, the process can beeasily automated and scaled-up using an off-the-shelf robot or ink-jetprinting instrument. Standard covalent attachment chemistries requirethe use of photolithographic and laser patterning techniques whichrequire multiple masking and lifting steps for high density DNA arraypreparation.

Third, unlike traditional techniques, the present method does notrequire the use of expensive crosslinking agents. These crosslinkingagents are difficult to use because of their sensitivity to air andhumidity. Therefore, the present method provides a new, efficient andinexpensive method for DNA array preparation, and particularly for largescale DNA array preparation.

In the preferred embodiment, this invention describes a method forimmobilizing nucleic acids on silane coated solid phase surfaces whichis useful in genetic analysis and other screening applications such asprotein-DNA binding assays. The invention pertains to oligonucleotidearrays comprising silane coated solid phase surfaces and unmodified ormodified oligonucleotides covalently attached to the silane coated solidphase, such arrays being useful for genetic analyses and combinatorialanalyses involving nucleic acids or proteins. The features of the coatedsurface enable standardized patterning of unique oligunucleotides ontosilane surface coatings.

In the most preferred embodiment, the invention provides for methods ofcovalent attachment of unmodified oligonucleotides ontomercapto-silanized surface or epoxy-silanized surfaces with high densityand high stability. The ease of preparation of unmodifiedoligonucleotides coupled with stable ether (epoxy) or thio-ether(mercapto) linkage attachments renders this method the most costeffective, with little or no variation in terms of the quality ofoligonucleotides, stability of attachment linkage and consistency inlarge scale batch to batch manufactures. Additionally, the hydrophobicproperty of silane surfaces also allows simultaneous patterning ofmultiple DNA probes in a high density and in a variety of array formats.Furthermore, a DNA array that is stable to high salt and denaturingconditions such as DMF, urea and elevated temperatures, has wide uses inminiaturized biotechniques such as genetic testing, sequencing byhybridization and combinatory selection of DNA binding molecules.

The covalent attachment of the present invention can be distinguishedfrom other means of attachment, such as van der Waals interaction andion-ion interactions. Thus, unlike other attachment means, thecovalently immobilized oligonucleotide will not be released from thesolid-phase during subsequent wash steps. The covalent attachmentgenerally provides more stable binding than noncovalent attachment underelevated temperatures and upon other chemical treatment; thus, givingmore flexibility for use in biochemical processes.

II. The Immobilization of Nucleic Acid Molecules Using Epoxy Chemistry

In a preferred embodiment of the present invention, a selective, highlyefficient method is provided which employs an epoxy-based attachmentchemistry to covalently attach nucleic acid molecules in an endselective manner to a solid-phase. Oligonucleotides have two freehydroxyl groups at the 5'- and 3'- ends which allow oligonucleotides toundergo chemical and/or enzymatic elongation, ligation andcircularization. The differences in steric hindrances between these twoend hydroxyl groups have enabled 5'-selective esterification,5'-selective tritylation and 5'-selective oxidation in controlledconditions. Unmodified oligonucleotides for attachment to solid phasesunder certain controlled conditions thus have the potential for the5'--OH to react preferentially over the 3'--OH towards epoxy activatedsurfaces. End-selective attachment is achieved in epoxy chemistry bytaking advantage of differences in steric hindrance between the 5' and3' end of the oligonucleotide. Accordingly, one could block eitherterminus (via phosphorylation, etc.) and therefore obtain adirectionally oriented attachment between the solid phase and theunblocked terminus.

The covalent bond described in the epoxy-based preferred embodiment ofthe present invention is a covalent ether linkage. End selectiveattachment ensures that the full sequence of the immobilizedoligonucleotide is accessible for any desired biochemical reaction.Since there is no need for any modification of oligonucleotides, thisembodiment dramatically reduces the cost and variation in terms of thequality of oligonucleotides. The epoxy-based attachment chemistry allowsattachment of all forms of DNA including pcr products or genomic DNA tothe silanized surface. Furthermore, in the case of epoxy-basedattachment chemistry, the chemical bonds between the silane layer andoligonucleotides are covalent ether linkages, which are stable to heat,high salt, and elevated temperatures.

III. The Immobilization of Nucleic Acid Molecules Using MercaptoChemistry

The present invention describes in another preferred embodiment arandom, highly efficient method which employs a mercapto-basedattachment chemistry to covalently attach nucleic acid molecules in anon-specific manner to a solid-phase. The covalent bond described inthis second preferred embodiment of the present invention is a covalentthioether linkage. Because this embodiment relies on non-specificbinding, the full sequence of the immobilized oligonucleotide may not beaccessible for all desired biochemical reactions. Since the mercaptogroup is very reactive in terms of radical reactions and easilydeionized under lower pH (pH 9), this allows a variety of reactions tooccur with nucleic acids. Heterocyclic purines (electron rich systemstabilizing radicals, particularly at position 7 of purines) andpyrimidines (electron deficient, a nucleophile acceptor) inoligonucleotides are good acceptors for either nucleophilic attack orradical reactions. In mercapto chemistry the highly reactive mercaptogroups allow mild conditions for attachment. The resulting array canundergo a variety of biochemical reactions and allows hybridization withhigh efficiency. The mercapto-based attachment chemistry allowsattachment of all forms of DNA including pcr products or genomic DNA tothe silanized surface. Furthermore, in the case of mercapto-basedattachment chemistry, the chemical bonds between the silane layer andoligonucleotides are covalent thioether linkages, which are stable toheat, high salt, and elevated temperatures.

The unmodified nucleic acid molecules, described in the presentinvention, may be either genomic DNA (i.e., DNA containing anon-translated region), cDNA (i.e., DNA lacking non-translated regions)or RNA; the nucleic acid molecule may also be either single or doublestranded. While any unmodified nucleic acid molecule may be immobilizedusing the present invention, the preferred nucleic acid molecule of thepresent invention is an unmodified single-stranded syntheticoligonucleotide. The method for making a synthetic oligonucleotide hasbeen previously described by Gait, M. J. (Oligonucleotide Synthesis APractical Approach, IRL Press Ltd., Oxford (1984)) and Sinha, N. D. etal. (Nucl. Acids Res. 12:4539-4557 (1984)) (herein incorporated byreference).

Synthesis of unmodified oligonucleotides of about 10 to about 250nucleotides in length may be performed on an ABI 392 DNA/RNA synthesizeraccording to phosphoramidite chemistry.

After synthesis, the unmodified oligonucleotides can be purified (forexample, using an HPLC column) to separate the full-lengtholigonucleotides from any contaminating prematurely terminated (i.e.,shortened) oligonucleotides. Prior to use in the coupling reaction, theoligonucleotides are concentrated, and, if desired, the molarconcentration of the oligonucleotides can be determined.

Although any of a variety of glass or plastic solid supports can be usedin accordance with the methods of the present invention, glass is thepreferred support. The support can be fashioned as a bead, dipstick,test tube, pin column, etc. However, an especially preferred support isa glass slide. Alternatively, the solid support can be a form ofpolystyrene plastic (e.g., 96-well microtiter plate, etc.).

Many different mercaptosilane compounds such as3-mercapto-propyltrimethoxy-silane, 3-mercaptopropyltriethoxysilane,(mercapto-methyl) dimethoxysilane and(mercaptoethyl)ethyldimethoxysilane, etc. can be used in the presentinvention for coating the solid support with sulfhydryl groups. Thegeneral formula for a mercaptosilane that can be used in this inventionis:

    HS(CH.sub.2)n--SiX.sub.3

wherein X is a hydrolyzable group such as alkoxy, acyloxy, amine orhalide, etc. All of the mercaptosilanes mentioned above are commerciallyavailable from United Chemical, Inc. or Aldrich Chemical Company, Inc.

The silane can be coated onto the solid-phase by any of a number ofmeans. For example, the mercaptosilane can be deposited onto the solidsurface as an aerosol or a vapor. Alternatively, the silane can bespread onto the solid-surface by mechanical means (e.g., a spreader bar,a saturated cloth, etc.).

An important feature of the present invention is the hydrophobic natureof silanes. Because of this feature, it is possible for an aqueoussolution to form extremely well defined beads on the surface of anysolid support coated with mercaptosilane. With an automated deliverysystem, such as a Hamilton robot or ink-jet printing method, it ispossible to form a very complex array of oligonucleotide probes on amercaptosilane coated glass slide. Such methods can deliver nano topico-liter size droplets with sub-millimeter spacing. Because theaqueous beads are extremely well defined, it is possible to create anarray with an extremely high density of oligonucleotide probes. Thus, itis possible to create arrays having greater than about 10,000 probedroplets/cm².

IV. The Use of Immobilized Nucleic Acid Molecules

Immobilized nucleic acid molecules, and more preferably, immobilizedoligonucleotides, make an ideal diagnostic tool. Specifically, theirversatility and simplicity make them ideal diagnostic tools for thedetection of infectious and genetic diseases, mutation analysis, etc.

Although the manner in which the nucleic acid molecules are immobilizedto the solid support can be random, one of the preferred embodiments ofthe invention is to arrange the nucleic acid molecules into an orderedarray. As used herein, an array is an orderly arrangement of nucleicacid molecules, as in a matrix of rows and columns. The chemistry of thepresent invention is such that an individual array can contain either afinite or an infinite number of unique immobilized nucleic acidmolecules.

There are two preferred methods to make a nucleic acid array: one is tosynthesize the specific oligonucleotide sequences directly onto thesolid-phase in the desired pattern (Southern, et al., Nucl. Acids Res.22:1368-1373 (1994); Maskos, et al., Nucl. Acids Res. 20:1679-1684(1992); and Pease, et al., Proc. Natl. Aced. Sci. 91:5022-5026 (1994);all of which are herein incorporated by reference) and the other is topre-synthesize the oligonucleotides on an automated DNA synthesizer(such as an ABI 392 and then attach the oligonucleotides onto thesolid-phase at specific locations (Lamture, et al., Nucl. Acids Res.22:2121-2125 (1994) and (Smith, et al., Nucl. Acids Res. 22:5456-5465(1994) both herein are incorporated by reference). In the first method,the efficiency of the coupling step of each base will affect the qualityand integrity of the nucleic acid molecule array. This method generallyyields a large percentage of undesired incomplete (shortened) sequenceswhich can create problems in the analysis step and effect the integrityof the analysis. Thus, the quality and integrity of an array synthesizedaccording to the first method is inversely proportional to the length ofthe nucleic acid molecule. Specifically, the synthesis of longeroligonucleotides results in a higher percentage of incomplete, shortenedsequences.

A second, more preferred, method for nucleic acid array synthesisutilizes an automated DNA synthesizer for DNA synthesis. The controlledchemistry of an automated DNA synthesizer allows for the synthesis oflonger, higher quality DNA molecules than is possible with the firstmethod. Also, the nucleic acid molecules synthesized according to thesecond method can be purified prior to the coupling step. Therefore, thequality of the nucleic acid molecule array can be expected to be muchhigher than the quality of the nucleic acid array of the first method.However, a simple, effective and specific oligonucleotide couplingchemistry is lacking for the attachment of presynthesizedoligonucleotides. The present invention describes a simple, effectiveand efficient method for coupling a pre-synthesized unmodifiedoligonucleotide onto a solid-phase by means of either an ether orthioether covalent linkage.

A. Hybridization Detection Of PCR Products

Thus, for example, covalently immobilized nucleic acid molecules may beused to detect specific PCR products by hybridization where the captureprobe is immobilized on the solid phase (Ranki et al, Gene 21: 77-85(1983); Keller et al., J. Clin. Microbiol. 29: 638-641 (1991); Urdea etal., Gene 61: 253-264 (1987). A preferred method would be to prepare asingle-stranded PCR product before hybridization. A sample, suspected tocontain the target molecule, or an amplification product thereof, wouldthen be exposed to the solid-surface and permitted to hybridize to thebound oligonucleotide.

The methods of the present invention do not require that the targetnucleic acid contain only one of its natural two strands. Thus, themethods of the present invention may be practiced on eitherdouble-stranded DNA, or on single-stranded DNA obtained by, for example,alkali treatment of native DNA. The presence of the unused(non-template) strand does not affect the reaction.

Where desired, however, any of a variety of methods can be used toeliminate one of the two natural stands of the target DNA molecule fromthe reaction. Single-stranded DNA molecules may be produced using thesingle-stranded DNA bacteriophage M13 (Messing, J. et al., Meth.Enzymol. 101:20 (1983); see also, Sambrook, J., et al. (In: MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)).

Several alternative methods can be used to generate single-stranded DNAmolecules. Gyllensten, U. et al., (Proc. Natl. Acad. Sci. (U.S.A.)85:7652-7656 (1988) and Mihovilovic, M. et al., (BioTechniques 7:14(1989)) describe a method, termed "asymmetric PCR," in which thestandard "PCR" method is conducted using primers that are present indifferent molar concentrations. Higuchi, R. G. et al. (Nucleic AcidsRes. 17:5865 (1985)) exemplifies an additional method for generatingsingle-stranded amplification products. The method entailsphosphorylating the 5'-terminus of one strand of a double-strandedamplification product, and then permitting a 5'→3' exonuclease (such asT7 gene exonuclease) to preferentially degrade the phosphorylatedstrand.

Other methods have also exploited the nuclease resistant properties ofphosphorothioate derivatives in order to generate single-stranded DNAmolecules (Benkovic et al, U.S. Pat. No. 4,521,509; Jun. 4, 1985);Sayers, J. R. et al. (Nucl. Acids Res. 16:791-802 (1988); Eckstein, F.et al., Biochemistry 15:1685-1691 (1976); Ott, J. et al., Biochemistry26:8237-8241 (1987)).

Most preferably, such single-stranded molecules will be produced usingthe methods described by Nikiforov, T. (commonly assigned U.S. Pat. No.5,518,900, herein incorporated by reference). In brief, these methodsemploy nuclease resistant nucleotide derivatives, and incorporate suchderivatives, by chemical synthesis or enzymatic means, into primermolecules, or their extension products, in place of naturally occurringnucleotides.

Suitable nucleotide derivatives include derivatives in which one or twoof the non-bridging oxygen molecules of the phosphate moiety of anucleotide has been replaced with a sulfur-containing group (especiallya phosphorothioate), an alkyl group (especially a methyl or ethyl alkylgroup), a nitrogen-containing group (especially an amine), and/or aselenium-containing group, etc. Phosphorothioate deoxyribonucleotide orribonucleotide derivatives are the most preferred nucleotidederivatives. Methods of producing and using such phosphorothioatederivatives are disclosed by Nikiforov, T. (U.S. Pat. No. 5,518,900).

B. Solid Phase DNA Sequencing

The methods of the present invention may also be used in the practice ofsolid-phase sequencing as described by Khrapko, K. R. et al. (DNA Seq.,1:375-388 (1991) and Drmanac, R. and Crkvenjakov, R., Int. T. GenomeRes., 1: 1-11 (1992)), both herein are incorporated by reference.

C. GBA™ Genetic Bit Analysis

The methods of the present invention may also be used to immobilize theoligonucleotides that are used in the GBA™ Genetic Bit Analysis (Goelet,P. et al., PCT Application No. 92/15712), herein incorporated byreference. GBA™ Genetic Bit Analysis a solid-phase method for the typingof single-nucleotide polymorphisms. Oligonucleotides having a definedsequence complementary to a region that lies immediately proximal ordistal to the variable nucleotide of a polymorphism would thus beprovided to a polystyrene microtiter well or glass plate, and incubatedwith a salt, in accordance with the above-described methods.

The immobilized primer is then incubated in the presence of a DNAmolecule (preferably a genomic DNA molecule) having a single nucleotidepolymorphism whose immediately 3'-distal sequence is complementary tothat of the immobilized primer. Preferably, such incubation occurs inthe complete absence of any dNTP (i.e. dATP, dCTP, dGTP, or dTTP), butonly in the presence of one or more chain terminating nucleotidederivatives (such as a dideoxynucleotide derivative), and underconditions sufficient to permit the incorporation of such a derivativeonto the 3'-terminus of the primer. As will be appreciated, where thepolymorphic site is such that only two or three alleles exist (such thatonly two or three species of ddNTPs, respectively, could be incorporatedinto the primer extension product), the presence of unusable nucleotidetriphosphate(s) in the reaction is immaterial. In consequence of theincubation, and the use of only chain terminating nucleotidederivatives, a single dideoxynucleotide is added to the 3'-terminus ofthe primer. The identity of that added nucleotide is determined by, andis complementary to, the nucleotide of the polymorphic site of thepolymorphism.

Using the method described in the present patent application,oligonucleotide primers can be immobilized on solid phases likepolystyrene or glass, hybridized to PCR-derived, single-strandedtemplates, and subjected to enzymatic extension at their 3'-ends by asingle, labeled ddNTP. The nature of the incorporated ddNTP isdetermined by the nucleotide that is located in the opposite strand (thepolymorphic nucleotide). This assay can be conveniently carried out bothin polystyrene ELISA plates, or on glass slides.

In this embodiment, the nucleotide of the polymorphic site is thusdetermined by assaying which of the set of labeled nucleotides has beenincorporated onto the 3'-terminus of the bound oligonucleotide by aprimer-dependent polymerase. Most preferably, where multipledideoxynucleotide derivatives are simultaneously employed, differentlabels will be used to permit the differential determination of theidentity of the incorporated dideoxynucleotide derivative.

D. Ligase-Mediated GBA™

The methods and reagents of the present invention can also be used inconcert with a polymerase/ligase mediated polymorphic interrogationassay. This assay, termed ligase-mediated GBA™ genetic bit analysis, isa more specific version of the GBA™ genetic bit analysis assay. Theadditional specificity arises from the addition of a secondhybridization step and a ligation step.

In this assay, two oligonucleotides are employed. The firstoligonucleotide is a primer that is complementary to the immediately3'-distal invariant sequence of the polymorphism. The 3'-end of theoligonucleotide is attached to the plate. A second linkeroligonucleotide is complementary to the 5'-proximal sequence of thepolymorphism being analyzed, but is incapable of hybridizing to thefirst oligonucleotide. The second linker oligonucleotide isphosphorylated at both its 3' and 5' ends.

These oligonucleotides are incubated in the presence of DNA containingthe single nucleotide polymorphism that is to be analyzed, and at leastone 2'-deoxynucleotide 5'-triphosphate. The incubation reaction furtherincludes a DNA polymerase and a DNA ligase. The tethered and solubleoligonucleotides are thus capable of hybridizing to the same strand ofthe target molecule under analysis. The sequence considerations causethe two oligonucleotides to hybridize to the proximal and distalsequences of the single nucleotide polymorphism (SNP) that flank thevariable nucleotide of the polymorphism, and to be separated by a singlenucleotide at the precise position of the variability.

The presence of a polymerase and the 2'-deoxynucleotide 5'-triphosphatecomplementary to the nucleotide present in the variable site of thepolymorphism permits the extended primer to be ligated to the boundoligonucleotide, thereby immobilizing the primer. The identity of thepolymorphic site that was opposite the single nucleotide can then bedetermined by any of several means. In a preferred embodiment, the2'-deoxynucleotide 5'-triphosphate of the reaction is labeled, and itsdetection thus reveals the identity of the complementary nucleotide ofthe polymorphic site. Several different 2'-deoxynucleotide5'-triphosphates may be present, each differentially labeled.Alternatively, separate reactions can be conducted, each with adifferent 2'-deoxynucleotide 5'-triphosphate. In an alternativesub-embodiment, the 2'-deoxynucleotide 5'-triphosphates are unlabeled,and the soluble oligonucleotide is labeled. In this embodiment, theprimer that is extended is immobilized on the polystyrene. Separatereactions are conducted, each using a different unlabeled2'-deoxynucleotide 5'-triphosphate.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein before set forth and as follows in the scopeof the appended claims.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention unless specified. All patents, patent applicationsand publications mentioned herein are incorporated by reference in theirentirety.

EXAMPLE 1 EPOXY-BASED CHEMISTRY

Attachment was obtained by a two-step process of silane treatment andoligonucleotide binding. Glass slides were etched in 25% aqueousammonium hydroxide, rinsed in milliQ water, then in 95% ethanol. Theywere treated for about 30 minutes in 3-glycidoxy propyl ethanol (95%ethanol, pH 4.5). Slides were cured at 65° C. for at least 12 hours.2.5-10 uM conc. of oligonucleotides in an alkaline solution (12.5 mmNaOH or KOH) were applied onto cured slides in any desired arrayformats, in a covered chamber overnight, after which they were stored inwater or a covered chamber for later use. A functional test withhybridization and enzymatic reactions gave the desired signal with 400>1signal to noise ratio by ELF indirect detection reading and radioisotopequantitation (FIG. 1). FIG. 1 shows the results of GBA functional assayof epoxy-based attachment chemistry. The 25-mer primer with (T)10 spacerarm at 5' was used for attachment. Standard GBA conditions were used interms of nucleoside triphosphate concentration and enzyme concentrationand reaction conditions. Synthetic template 1 was designed to give an"A" signal and synthetic template 2 was designed to give a "T" signal.Single base extension (GBA signal) was obtained only with appropriatetemplate, by using ELF indirect detection and also by CCD cameraimaging. The signal to noise ratio (S/N) was greater than 400:1.

Additionally, an attachment experiment was designed with the followingend-blocked oligonucleotides 5'-end blocked, 3'-end blocked, or both endblocked. The end blocks were achieved by phosphorylation of the endhydroxyl groups during automated DNA synthesis. The attachment for eachtype of oligonucleotides was quantified by phosphor image analysis andthen the ratio of end selectivity was determined. The 5'-end selectiveattachment of unmodified oligonucleotides to epoxy silanized surfaceswas demonstrated with a selectivity ratio of at least 5:1. Attachmentthrough heterocyclic aminos was minimal in certain conditions tested(Table 1).

                  TABLE 1    ______________________________________    Oligo conc. used for att. (uM)                     10      5       2.5   1.25    5'-att. (incl. middle)pmole/7mm2                     1.0     1.6     1.4   0.9    3'-att. (incl.   0.143   0.29    0.42  0.48    middle)pmoles/7mm2    middle att. pmole/7mm2                     0.13    0.09    0.35  0.235    5'-att./3'-att.(after sub middle)                     87      7.55    15    21    5'-att./middle att.                     6.7     16      3     2.8    3'att./middle att.                     0.077   2.2     0.2   1.04    3'-& 5-att./middle att.                     6.8     19      3.2   3.8    ______________________________________

Table 1 shows the end selective attachment of un-modifiedoligonucleotides by epoxy-based chemistry. Attachment was performed onan epoxy silanized slide with various concentrations of oligonucleotidesin 12.5 mM NaOH for overnight and washed sequentially with TNTw and 50mM NaOH. 5'-att refers to the attachment reading of 3'-phosphorylatedoligonucleotides; 3'-att refers to the attachment reading of5'-phosphorylated oligonucleotides. Middle att refers to theheterocyclic amino attachment reading of both 3'- and 5'-end blockedoligonucleotides. The ³² pisotopes images were analyzed on phosphorimage quanta software. Attachment at a concentration of 10 uM gave thebest selectivity ratio of 87:1 of 5'-end vs. 3'-end attachment.Attachment at concentration of 5 uM gave the best end selectivity ratioof 19:1 of 3'-plus 5'-vs. middle attachment. These results were based onone slide only, however, a number of slides expressed a trend of optimumconcentration at 2.5-5 uM for 5'-end selective attachment. In addition,epoxy-silanized surfaces are air-, moisture-, and heat-stable and showedno nonspecific binding to proteins and oligonucleotides under conditionstested. In summary, 5'-end selective attachment of oligonucleotides wasdemonstrated without modification of oligonucleotides.

EXAMPLE 2 MERCAPTO-BASED CHEMISTRY

Attachment was obtained by a two-step process of silane treatment andoligonucleotide binding. Glass slides were etched in 25% aqueousammonium hydroxide, rinsed in milliQ water, then in 95% ethanol. Theywere treated for about 30 minutes in 3-mercapto-propyl-trimethoxysilane(MPTS). Slides were cured for at least 24 hours under dry inert gas (Aror N₂). 2.5-10 uM conc. of oligonucleotides in an alkaline solution wereapplied onto cured slides in any desired array formats, in a coveredchamber for an overnight, after which they were stored in water or acovered chamber for later use. A functional test with hybridization andenzymatic reactions gave the desired signal (FIG. 2). FIG. 2 shows theresults of mercapto-based attachment chemistry and functional assay byhybridization and GBA. The data was based on phosphor imaging quantaanalysis. The X axis represents the input concentration ofoligonucleotides used for attachment from 0.3125 uM to 40 uM. Oligoattached is represented by the triangle (pmole/7 mm²), the diamondrepresents hybridization and the square represents GBA signals (ddATPextension). ³² P labeled oligonucleotides were used for determining theattachment density. The ³² P 5'-template was used for assessing thehybridization efficiency. The GBA efficiency was determined byincorporation of ³² P-ddATP with exo-klenow DNA polymerase without thepresence of other cold dideoxynucleotides. The GBA efficiency andhybridization reached saturation at the attachment input of 10 uM.

Additionally, experiments were performed for attachment of dye-labelednucleotides to mercapto propyl silanized slides which showed strongersignals for A and G and weaker signals for C and T, which favors thehypothesis that radical mechanisms are more involved than nucleophilicattacks. The attachment of ³² P labeled oligonucleotides and fluoresceinlabeled oligonucleotides was demonstrated and quantified (FIG. 2). Theattachment results achieved in terms of attachment density,hybridization and genetic bit analysis efficiency were compatible withepoxy chemistry and chemistries reported by others.

EXAMPLE 3 THE RELATIONSHIP BETWEEN PRIMER DENSITY AND HYBRIDIZATIONEFFICIENCY

Hybridization efficiency is positively related to the surface density ofthe attached primer. In this study, increasing amounts of the BRAC1primer 5' (T)₁₀, TCA TTA ATG CTA TGC AGA AAA TCT TAG (SEQ ID No. 1) arecovalently attached to a solid surface according to the methodsdescribed above. Table 2 shows the results for epoxy-based attachmentvs. hybridization efficiency. The data was based on phosphor imagingquanta analysis. 3'-phosphorylated oligonucleotides were used forattachment and 5'-phosphorylated templates were used for hybridiation. Alow surface coverage would presumably yield a corresponding lowhybridization signal. Conversely, high surface densities might result insteric interference between the covalently immobilized oligonucleotidesthereby impeding access to target DNA. The results indicated that withhigher coverage up to 1.78 pmoles/7 mm², a higher hybridizationefficiency was obtained. Increased primer density is associated withincreased hybridization efficiency. Accordingly, hybridizationefficiency is affected by the stability of the primer attachment.

                  TABLE 2    ______________________________________    conc.for att. covalent att                            Hybridization    ______________________________________     40 uM        0.19       0.1 + 0.015    20            0.42      0.14 + 0.00    5             1.78       0.58 + 0.005    2.5           1.61      0.43 + 0.03     1.25         0.98      0.29 + 0.05      0.625       0.42      0.17 + 0.01    ______________________________________

EXAMPLE 4 GENETIC BIT ANALYSIS COMPATIBILITY

A GBA primer having a poly-T₁₀ residue long spacer arm is attached tothe glass surface by means of the previously described epoxy-basedchemistry or mercapto-based chemistry. Standard GBA biochemistry is usedto analyze two synthetic templates. Each synthetic template ishybridized to GBA primer immobilized to the treated glass slide andtreated with an extension mix containing all of the extension reactioncomponents, exonuclease-free Klenow fragment of the E. coli polymeraseand each of four fluorescein-labeled ddNTP's and co-ddNTP's or ³²P-ddATP. The signal was recorded by enzyme-mediated fluorescence using aCytoflour II fluorescent plate reader (FIG. 1) or phosphor image quantaanalysis. FIG. 1 shows the results of GBA functional assay ofepoxy-based attachment chemistry. The 27-mer primer with (T)10 spacerarm at 5' was used for attachment (SEQ ID No. 1). Standard GBAconditions were used in terms of nucleoside triphosphate concentrationand enzyme concentration and reaction conditions. Synthetic template 15' ACA CTC TAA GAT TTT CTG CAT AGC ATT AAT (SEQ ID No. 2) was designedto give an "A" signal and synthetic template 2 5' GGA CAC TAA GAT TTTCGT CAT AGC ATT AAT (SEQ ID No. 3) was designed to give a "T" signal.Single base extension (GBA signal) was obtained only with theappropriate template, by using ELF indirect detection and also by CCDcamera imaging. The signal to noise ratio (S/N) was greater than 400:1.

DNA Samples. Genomic DNA was isolated using the SDS/Proteinase Kprocedure (Maniatis, T. Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor (1989)) fromperipheral blood nucleated cells of humans or horses enriched from redblood cells by selective lysis accomplished by diluting blood with athree fold volume excess of ACK lysing buffer (0.15M ammonium chloride,1 mM potassium bicarbonate, 0.1 mM EDTA). Oligonucleotides were preparedby solid-phase phosphoramidite chemistry using an Applied Biosystems,Inc. Model 391 automated DNA synthesizer (Foster City, Calif.). In thecase of primers used in Genetic Bit Analysis (GBA) reactions,detritylation was not performed following the final cycle of synthesisand the full-length oligonucleotide was purified using the AppliedBiosystems oligonucleotide purification cartridge (OPC) as recommendedby the manufacturer. For most PCR reactions, primers were used directlyby drying down the de-protection reaction.

Table 3 depicts the results of an experiment employing a synthetictemplate 15' ACA CTC TAA GAT TTT CTG CAT AGC ATT ATT (SEQ ID No.2)(designed to give a GBA signal in base A) and a synthetic template 2,5' GGA CAC TAA GAT TTT CGT CAT AGC ATT AAT (SEQ ID No. 3) (designed togive a signal in base T). The primer used was (T)₁₀ TCA TTA ATG CTA TGCAGA AAA TCT TAG (SEQ ID No. 1). Both signals give strong signals in theexpected bases with virtually no noise observed in the other bases (theSignal to Noise Ratio ranged from 520:1 to 600:1).

                  TABLE 3    ______________________________________                  Fluorescent Counts    Nucleotide Inserted                    Template 1                              Template 2    ______________________________________    A               ≈600                              1    C               ND        ND    G               ND        ND    T               1         ≈520    ______________________________________

EXAMPLE 5 PREPARATION OF AN OLIGONUCLEOTIDE ARRAY BY INK-JET PRINTING

Another method for the automated delivery of the oligonucleotidesolution employs an ink-jet printing technique performed by MicroFab(MicroFab Technologies, Inc., Plano, Tex.). In one experiment, fourdifferent spot spacings (center to center) and eight different dropletsizes are tested on the mercaptosilane coated surface using anoligonucleotide labeled at the 3'-terminus with fluorescein. The formatof the slides, depicted in Table 4, are as follows:

                  TABLE 4    ______________________________________                     Row    Slide    Spot    (Row Spacing = 6 mm)    No.      Spacing Row 1       Row 2 Row 3    ______________________________________    Slide 1   1 mm    5 nl        10 nl                                       25 nl    Slide 2  0.5 mm   1 nl        2 nl 5 nl    Slide 3  250 μm                     250 pl      500 pl                                       1 nl    Slide 4  125 μm                     125 pl      250 pl                                       N/A    ______________________________________

The labeled oligonucleotides are detected using a Molecular DynamicFluorlmager 595. The ink-jet printing technique is a suitable method forthe manufacture of oligonucleotide arrays with sub-millimeter spacingand nano to pico-liter droplet sizes. As such, the ink-jet printingtechnique is suitable for large scale manufacture of oligo arrays.

EXAMPLE 6 PREPARATION OF AND OLIGONUCLEOTIDE ARRAY WITH AN AUTOMATICPIPETING ROBOT

A Hamilton 2200 automated pipeting robot is used to make arrays ofoligonucleotide drops, ranging in size from about 100 nl to about 250nl, with 1 mm spacing between dots. The small volumes of oligonucleotidesolution used with the automated pipeting robot allows for rapid dryingof the oligonucleotide drops. As in the ink-jet printing method, aHamilton robot can be programmed to deliver nano to pico-liter sizedroplets with sub-millimeter spacing.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein before set forth and as follows in the scopeof the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 3    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 37 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #      37          ATGC TATGCAGAAA ATCTTAG    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #           30     TGCA TAGCATTATT    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #           30     GTCA TAGCATTAAT    __________________________________________________________________________

What is claimed is:
 1. A method for immobilizing an unmodified nucleic acid molecule to a glass or plastic solid phase, which comprises the steps of:(A) coating said solid phase with a mercapto-silane or an epoxy-silane and allowing said silane-coated solid phase to cure; and (B) coupling said unmodified nucleic acid molecule having either a terminal 3' OH or a terminal 5' OH to said silane-coated solid phase.
 2. The method of claim 1, wherein in step A, said coating occurs in the presence of an acidic buffer of aqueous ethanol for 30 minutes.
 3. The method of claim 1, wherein in step A, said silane is selected from the group consisting of mercapto-alkyl-trimethoxysilane and glycidoxyalkyl-silane.
 4. The method of claim 1, wherein in step A, said silane is 3-mercaptopropyl-trimethoxysilane.
 5. The method of claim 4, wherein in step A, said curing occurs for at least 12 hours in the presence of a dry inert gas consisting of Ar or N₂.
 6. The method of claim 4, wherein in step B, said covalent linkage is a covalent thioether linkage.
 7. The method of claim 1, wherein in step A, said silane is 3-glycidoxy trimethoxypropyl silane.
 8. The method of claim 7, wherein in step A, said curing occurs at a temperature of from 60° C. to 70° C. for 10-14 hours.
 9. The method of claim 7, wherein in step B, said covalent linkage is a covalent ether linkage.
 10. The method of claim 1, wherein in step B, said coupling occurs in alkaline solution.
 11. The method of claim 10, wherein in step B, said alkaline solution comprises from 0.0001M to 5M NaOH.
 12. The method of claim 10, wherein in step B, said alkaline solution comprises NaOH, KOH, and LiOH.
 13. The method of claim 1, wherein in step B, said nucleic acid molecules comprise oligonucleotides.
 14. The method of claim 13, wherein said oligonucleotides have a concentration of from 1.0 to 10 μM.
 15. The method of claim 1, wherein in step B, said covalent linkage is selected from the group consisting of covalent ether and thioether linkages.
 16. The method of claim 1, wherein said solid phase is glass.
 17. The method of claim 16, wherein said glass is a microscope slide.
 18. The method of claim 1, wherein said solid-phase is plastic.
 19. The method of claim 18, wherein said plastic is polystyrene plastic.
 20. The method of claim 19, wherein said polystyrene support is a microwell plate.
 21. Ache method of claim 19, wherein said polystyrene support is an array designed to fit into a microwell plate.
 22. The method of claim 1, wherein said solid-phase is selected from the group consisting of a bead, a plate, a column, a pin and a dipstick.
 23. The method of claim 1, wherein the coupling reaction creates an array of immobilized nucleic acid molecules.
 24. The method of claim 1, wherein said immobilized nucleic acid is a polynucleotide and wherein said method optionally further comprises the steps of:(C) capturing from a solution at least one strand of a specific polynucleotide analyte by hybridization to said immobilized polynucleotide; and (D) detecting the presence of the captured analyte.
 25. The method of claim 24, wherein step C further comprises the steps of:(C'(1)) amplifying a specific region of a specific genome using a polymerase chain reaction, said region having a sequence complementary to said immobilized polynucleotide; and (C'(2)) capturing from solution at least one strand of said amplification product by hybridization to said immobilized polynucleotide; and wherein step D further comprises the step of (D') detecting the presence of the captured amplification product.
 26. The method of claim 25, wherein said method optionally further comprises the steps of:(E) incubating a sample of nucleic acid of a target organism, containing a single nucleotide polymorphism in the presence of said immobilized polynucleotide primer and at least one dideoxynucleotide derivative, under conditions sufficient to permit a polymerase mediated, template-dependent extension of said primer, said extension causing the incorporation of a single dideoxynucleotide to the 3'-terminus of said primer, said single dideoxynucleotide being complementary to the single nucleotide of the polymorphic site of said polymorphism; (F) permitting said template-dependent extension of said primer molecule, and said incorporation of said single dideoxynucleotide; and (G) determining the identity of the nucleotide incorporated into said polymorphic site, said identified nucleotide being complementary to said nucleotide of said polymorphic site.
 27. The method of claim 1, wherein said coating step is by means of an aerosol, a vaporization means or any other mechanical means.
 28. A method for immobilizing an unmodified nucleic acid molecule to a mercapto-silane or epoxy-silane-coated solid phase comprising coupling said unmodified nucleic acid molecule, wherein said nucleic acid molecule has either a terminal 3' OH or a terminal 5' OH, to said silane-coated solid phase.
 29. The method of claim 28, wherein said silane is selected from the group consisting of mercapto-alkyl-trimethoxysilane, glycidoxy-alkyl-silane, 3-mercapto-propyl-trimethoxysilane, and 3-glycidoxy propyl trimethoxysilane. 